History of RNAi

RNA interference (RNAi) was first identified in C. elegans by Nobel laureates Fire and Mello (1), and now represents one of the most promising discoveries in molecular biology. Endogenous RNAi activity has been linked to the regulation of transposon mobility (2), the determination of gene expression profiles (3) and cell fate (4), and is a crucial component of the innate cellular defense against viral infection in vivo(5). Three unique RNAi mechanisms controlling target gene expression have been demonstrated. RNAi regulates gene transcription by modifying heterochromatin formation (6). RNAi exercises two forms of post-transcriptional control. First RNAi can inhibit the translation of target mRNA (7) and second, RNAi can direct target mRNA destruction through the RISC complex (8). DICER first processes dsRNA leaving a two nucleotide long 3' overhang. This primes the dsRNA for binding to the RISC complex and leads to the activation of the enzyme activity of argonaute, the RNAse component of the RISC complex that destroys one of the RNA strands. The remaining guide strand, through complementary binding, then leads the RISC complex to associate with and cleave target RNA molecules.

The discovery of RNAi introduced an extraordinarily powerful laboratory tool for researchers and became a promising potential therapeutic tool, consequently leading to the 2006 Nobel Prize in Physiology or Medicine being awarded to Andrew Z. Fire and Craig C. Mello. In the laboratory, RNAi molecules are being used to downregulate individual target gene expression in a variety of organisms and cell types, exploiting each of the three mechanisms of inhibiting gene expression described above. These techniques are useful for manipulating an experimental system to explore individual gene and protein functions as well as their relationships to other genes and proteins. RNAi also has exciting clinical potential (9).

Details of these RNAi mechanisms are popular subjects of rigorous study, though much remains to be clarified. RNAi control of target mRNA degradation through the RISC complex, however, is the most well-described as well as the intended mechanism for RNAi Gene Silencers.

Santa Cruz Biotechnology, Inc. offers a complete line of RNAi Gene Silencers, including siRNA, shRNA Plasmid and shRNA Lentiviral products covering > 99% of human and mouse protein encoding genes.

RNAi-directed mRNA Cleavage

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siRNA Gene Silencers shRNA Plasmids shRNA Lentiviral Particles Frequently Asked Questions Back to top

siRNA Gene Silencers

• siRNA refers to small interfering or short interfering RNA
• Requires transfection of cells using a lipid-based transfection reagent
• Useful for a transient knock-down

• siRNA Gene Silencers are pools of three target specific 19-25 nucleotide-long double stranded RNA molecules with 2-nt 3' overhangs on each end
• 10 µM, 50-100 transfections
• for independent verification of target gene silencing results, individual siRNA duplex components are also available upon request

• suitable control antibodies are available
• RT-PCR Primers are available
• siRNA Dilution Buffer, sc-29527
• siRNA Transfection Reagent, sc-29528
• siRNA Transfection Medium, sc-36868
• siRNA Reagent System, sc-45064
• Control siRNAs, including Control siRNA-A, sc-37007
• Control siRNA (FITC Conjugate)-A, sc-36869

How do siRNA Gene Silencers work?

shRNA Plasmid Gene Silencers

• shRNA refers to small hairpin or short hairpin RNA
• Plasmids encoding shRNA enter the cell via lipid-based transfection
• shRNA plasmids are capable of transient or stable inhibition of target gene expression
• shRNA Plasmids are provided as a pool of three to five lentiviral vector plasmids which each encode a target specific 19-25 nt shRNA with a 6 bp loop
• 20 µg, up to 20 transfections
• shRNA transcription is under the control of the H1 promoter
• provided as transfection-ready purified plasmid DNA
• After transfection, cells stably expressing shRNA can be selected by puromycin treatment

• suitable control antibodies are available
• RT-PCR Primers are available
• shRNA Plasmid Transfection Reagent, sc-108061
• shRNA Plasmid Transfection Medium, sc-108062
• Control shRNA Plasmid-A, sc-108060
• Control shRNA Plasmid-B, sc-108065
• Control shRNA Plasmid-C, sc-108066

Confirm shRNA Plasmid Gene Silencer transfection efficiency with copGFP Control Plasmid: sc-108083

Generate Cells with stable expression of shRNA

How do shRNA Plasmid Gene Silencers work?

shRNA Lentiviral Particles

• shRNA refers to small hairpin or short hairpin RNA
• Lentiviral Particles deliver a shRNA encoding plasmid to target cell
• Useful for either transient or stable knock-down of a target gene
• Lentiviral Particles are provided as transduction-ready viruses for targeted gene silencing in mammalian cells (human or mouse)
• 200 µl frozen viral stock containing 10⁶ infectious units of virus (IFU), sufficient for 10-20 transductions
• The Lentiviral Particles generally contain three to five expression constructs, each construct encoding a target specific 19-25 nt shRNA with a 6 bp loop
• After transduction, cells stably expressing shRNA can be selected by puromycin treatment
• copGFP Control Lentiviral Particles allow confirmation of the transduction efficiency of the Lentiviral Particles in a target cell population by expression of GFP detectable by either flow cytometry or fluorescence microscopy.
• The benefits of using shRNA Lentiviral Particles include avoiding harsh transfection techniques and the ability to introduce shRNA to any cell type
• Biosafety information - Lentiviral Particles are replication-incompetent and are designed to self-inactivate after transduction and integration of shRNA constructs into the genomic DNA of target cells.

• suitable control antibodies are available
• RT-PCR Primers are available
• Control shRNA Lentiviral Particles: sc-108080
• copGFP Control Lentiviral Particles: sc-108084
• Puromycin dihydrochloride: sc-108071

Generate Cells with stable expression of shRNA

How do Lentiviral Particle Gene Silencers work?

What are the advantages of using shRNA versus siRNA?

Transfection of siRNA Gene Silencers into cultured cells provides a fast and efficient, though short-term, decrease in target gene expression. One may achieve stable gene silencing using shRNA Plasmids or shRNA Lentiviral Particles followed by puromycin selection. So, if one is targeting the expression of a protein with slow turnover, shRNA Plasmid or shRNA Lentiviral Particles would be ideal for accomplishing the goal.

What is the difference between using shRNA Lentiviral Particles versus shRNA Plasmids?

Transfection is required to use shRNA Plasmids for target gene silencing. Whereas shRNA Lentiviral Particles arrive ready to add to virtually any mammalian cell type, including primary and non-dividing cells. Both shRNA Plasmids and shRNA Lentiviral Particles may be used to develop stable expression of the shRNA with puromycin treatment. Lentiviral particles are shipped on dry ice while shRNA Plasmids are shipped on blue ice.

Do Lentiviral shRNA products pose any safety concerns?

Lentiviral particles can be employed in standard Biosafety Level 2 tissue culture facilities (and should be treated with the same level of caution as with any other potentially infectious reagent). The Lentiviral Particles are replication-incompetent and are designed to self-inactivate after transduction and integration of the shRNA constructs into the genomic DNA of target cells.

Are the sequences of your shRNA products the same as those for your related siRNA products to the same gene? Do you make those sequences available?

Yes. The sequences encoded in our shRNA Plasmids are the same as those used in the corresponding siRNA Gene Silencer products. These sequences are available to customers. Contact your Technical Service Representative.

The shRNA Plasmids are provided as a pool of three to five plasmids. Are they provided in separate vials? Are the individual shRNA plasmids of a pooled product sold separately?

The shRNA Plasmid products are provided in one vial. We offer the siRNA strands separately upon request. We may offer the plasmids separately in the future.

What kind of lentiviral vector do you use? What is the "vector name"?

The lentiviral vector we use is a custom made, proprietary vector. Please let us know what information you are looking for and why you need it. We might be able to answer your question without disclosing proprietary information.

What type of promoter does your vector use for shRNA transcription?

The vector uses a H1 promoter

What type of selection marker(s) are in the vector?

The vector has a Puromycin resistance gene encoding puromycin N-acetyltransferase enzyme for selection of successfully transfected or transduced cells.

How do you propagate the lentiviral vector plasmid?

The shRNA Plasmids and Lentiviral Particles are sold as transfection / transduction ready products. No additional preparation is necessary. shRNA Gene Silencers are consumable products for which no propagation protocols are provided.

What is copGFP and how is it helpful for use with the shRNA plasmids and Lentiviral Particles?

By administering the copGFP plasmid or copGFP Lentiviral Particles to a separate sample of target cells, one can identify the transfection or viral transduction efficiency for the target cell population. The copGFP plasmid and copGFP Lentiviral particles lead to expression of copepod green fluorescent protein which can be detected using a fluorescence microscope or flow cytometer.

What is the difference between (h) and (h2) shRNA products (for example E-Cadherin, sc-35242-SH and sc-44222-SH)?

The (h) and (h2) products are designed to silence the same gene, they have different sequences.

What support products and transfection reagents must I purchase from SCBT to use your shRNA Plasmids?

We recommend our shRNA Plasmid DNA Transfection Reagent, sc-108061 in addition to shRNA Plasmid DNA Transfection Medium, sc-108062. We also recommend our control shRNA Plasmid DNAs, either sc-108060 (A), sc-108065 (B) or sc-108066 (C). These encode scrambled shRNA sequences which will not target any known mammalian mRNA.

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References

1. Fire, A., Xu, S.Q., Montgomery, M.K., Kostas, S.K., Driver, S.E. and Mello, C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811.
2. Das, P.P., Bagijn M.P., Goldstein, L.D., Woolford, J.R., Lehrbach, N.J., Sapetschnig, A., Buhecha, H.R., Gilchrist, M.J., Howe, K.L., Stark, R., Matthews, N., Berezikov, E., Ketting, R.F., Tavaré, S. and Miska E.A. 2008. Piwi and piRNAs Act Upstream of an Endogenous siRNA Pathway to Suppress Tc3 Transposon Mobility in the Caenorhabditis elegans Germline. Mol Cell 31: 79-90.
3. Kawasaki, H., Taira, K. and Morris, K.V. 2005. siRNA Induced Transcriptional Gene Silencing in Mammalian Cells. Cell Cycle 4: 442-448.
4. Georgantas III, R.W., Hildreth, R., Morisot, S., Alder, J., Liu, C.G., Heimfeld, S., Calin, G.A., Croce, C.M. and Civin, C.I. 2007. CD34+ hematopoietic stem-progenitor cell microRNA expression and function: A circuit diagram of differentiation control. PNAS 104: 2750-2755.
5. Chotkowskia, H.L., Ciotab, A.T., Jiab, Y., Puig-Basagoitic, F., Kramerb, L.D., Shic, P.Y. and Glaser, R.L. 2008. West Nile virus infection of Drosophila melanogaster induces a protective RNAi response. Virology 377: 197-206.
6. Kawasaki, H., Taira, K. and Morris, K.V. 2005. siRNA Induced Transcriptional Gene Silencing in Mammalian Cells. Cell Cycle 4: 442-448.
7. Tamura, Y., Yoshida, M., Ohnishi, Y. and Hohjoh, H. 2008. Variation of gene silencing involving endogenous microRNA in mammalian cells. Mol Biol Rep, epub.
8. Hammond,S.M., Boettcher, S., Caudy, A.A., Kobayashi, R. and Hannon, G.J. 2001. Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi. Science 293: 1146-1150.
9. Zhanga, Y., Yanga, H., Xiaoa, B., Wub, M., Zhoua, W., Lia, J., Lib, G. and Christados, P. 2008. Dendritic cells transduced with lentiviral-mediated RelB-specific ShRNAs inhibit the development of experimental autoimmune myasthenia gravis. Mol Imunol, epub.